How to measure tumour response in rectal cancer? An explanation of discrepancies and suggestions for improvement

Open AccessPublished:January 17, 2020DOI:https://doi.org/10.1016/j.ctrv.2020.101964

      Highlights

      • Use of tumour regression grading is limited by lack of standardisation.
      • Tumour shrinkage as a response mechanism allows local excision as definite treatment.
      • Tumour fragmentation is associated with a relatively poor outcome.

      Abstract

      Various methods categorize tumour response after neoadjuvant therapy, including down-staging and tumour regression grading. Response categories allow comparison of different treatments within clinical trials and predict outcome. A reproducible response categorization could identify subgroups with high or low risk for the most appropriate subsequent treatments, like watch and wait. Lack of standardization and interpretation difficulties currently limit the usability of these approaches. In this review we describe these difficulties for the evaluation of chemoradiation in rectal cancer.
      An alternative approach of tumour response is based on patterns of residual disease, including fragmentation. We summarise the evidence behind this alternative method of response categorisation, which explains a number of very relevant clinical discrepancies. These issues include differences between downstaging and tumour regression, high local regrowth in advanced tumours during watchful waiting procedures, the importance of resection margins, the limited value of post-treatment biopsies and the relatively poor outcome of patients with a near complete pathological response. Recognition of these patterns of response can allow meaningful development of novel biomarkers in the future.

      Keywords

      Introduction

      In the 1990s preoperative radiotherapy and chemoradiation (CRT) were administered in a range of tumours with the aim of reducing tumour volumes to enhance operability prior to definitive surgical intervention. Attempts to show a distinction between responders and non-responders by grading the pathological effects of preoperative CRT treatment originated with Mandard with a model proposed in oesophageal cancer [
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      Pathologic assessment of tumor regression after preoperative chemoradiotherapy of esophageal carcinoma. Clinicopathologic correlations.
      ]. Using an arbitrary scale to describe the balance between residual fibrosis and residual viable tumour, the authors categorised the degree of pathological tumour regression into 5 grades. These grades were shown to have prognostic relevance, such that a major histopathological response was significantly associated with better disease-free survival. This system was then extrapolated to rectal cancer, where patients with a better response to preoperative treatment also appear to have a better prognosis [
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      ].
      In the present day, neoadjuvant treatment offers an in vivo individualized tumour sensitivity testing by allowing evaluation of tumour response and reaction to a specific systemic or localised therapy.
      This evaluation can be performed within days to weeks of the start of the intervention and prior to surgery by assessing volume effects using RECIST criteria with CT or MRI [
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      ], MRI based tumour regression grading [
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      ], and mapping macroscopic and microscopic details about tumour architecture [
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      The predictive value of pre-/postneoadjuvant chemoradiotherapy MRI characteristics for patient outcomes in locally advanced rectal cancer.
      ]. Results of prospective trials showed clinical complete response (cCR) or TNM down-staging after CRT is a biomarker of more favourable oncological outcomes in LARC [
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      ]. Other investigators have monitored metabolic changes (FDG-PET scanning) after short course preoperative radiotherapy and CRT. Significant reductions in metabolic activity following 5 × 5 Gy have been reported with a median SUVmax reduction of 39.3% at 7 days [
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      ]. Many attempts have been made to achieve concordance between the degree of response on imaging and the histopathological grading [
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      ].
      The increase in the use of neoadjuvant treatment of rectal cancer over the past two decades [
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      ,
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      • Zar N.
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      Time trends, improvements and national auditing of rectal cancer management over an 18-year period.
      ,
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      ] such that CRT represents the standard of care for locally advanced cancer has prompted further discussions regarding categorisation/identification of tumour response in relation to the possibilities of total neoadjuvant therapy and other forms of organ preserving treatment strategies and determining the optimal timing of surgery. A high concordance between radiologically-based predictions and pathological findings has led to the widespread use of high-resolution MRI in the preoperative assessment of rectal cancer [
      • Nahas S.C.
      • Nahas C.S.R.
      • Cama G.M.
      • de Azambuja R.L.
      • Horvat N.
      • Marques C.F.S.
      • et al.
      Diagnostic performance of magnetic resonance to assess treatment response after neoadjuvant therapy in patients with locally advanced rectal cancer.
      ]. MRI is widely used to identify accepted risk factors - such as a threat to, or a breach of the mesorectal fascia and the presence of extramural venous invasion (EMVI).
      Yet, the innate genetic heterogeneity of rectal cancer leads to a variety of treatment responses [
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      ,
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      ] that can be recognised either with high resolution imaging techniques or in histological slides. Imaging does not require assessment of the resection specimens and hence can be repeated in vivo and compared sequentially. However, histology is the gold standard, especially since MRI cannot reliably distinguish complete response from near-complete response with extensive remaining fibrosis after treatment [
      • Nahas S.C.
      • Nahas C.S.R.
      • Cama G.M.
      • de Azambuja R.L.
      • Horvat N.
      • Marques C.F.S.
      • et al.
      Diagnostic performance of magnetic resonance to assess treatment response after neoadjuvant therapy in patients with locally advanced rectal cancer.
      ]. In general, two approaches are applied for quantification of this response, with their own advantages and disadvantages: tumour downstaging and tumour regression grading (TRG). Theoretically, these approaches would allow for comparisons between cohorts and trials, and might even be used as surrogate endpoints in neoadjuvant clinical trials. Unfortunately, there is a lack of standardization and a wide variation between different studies with dissimilar scales and groupings leading to diverse outcomes. There are also considerable subjective interpretation and inter-observer inconsistencies, all of which hamper implementation.
      A complicating factor is the timing of response assessment. Population studies [
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      ] as well as clinical trials that did not randomise for treatment intervals [
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      ] suggest that longer intervals between RCT and surgery results in more profound responses. This implies that even after 8 weeks there is a continuation of tumour regression and that timing is indeed an essential modulator of the reporting of responses. Unfortunately, there is a huge variation in timing within and between different studies, which makes comparisons very difficult. Moreover, results from randomised clinical trials focussing on this interval are conflicting [
      • Akgun E.
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      Randomized clinical trial of short or long interval between neoadjuvant chemoradiotherapy and surgery for rectal cancer.
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      Randomized controlled trial of 8 weeks' vs 12 weeks' interval between neoadjuvant chemoradiotherapy and surgery for locally advanced rectal cancer.
      ]. When comparing 7 versus 11 weeks interval, there was no difference in complete response rates, tumour stage or TRG in a French study including 265 rectal cancer patients [
      • Lefevre J.H.
      • Mineur L.
      • Kotti S.
      • Rullier E.
      • Rouanet P.
      • de Chaisemartin C.
      • et al.
      Effect of interval (7 or 11 weeks) between neoadjuvant radiochemotherapy and surgery on complete pathologic response in rectal cancer: a multicenter, randomized, controlled trial (GRECCAR-6).
      ]. In two Turkish randomised trials [
      • Akgun E.
      • Caliskan C.
      • Bozbiyik O.
      • Yoldas T.
      • Sezak M.
      • Ozkok S.
      • et al.
      Randomized clinical trial of short or long interval between neoadjuvant chemoradiotherapy and surgery for rectal cancer.
      ,
      • Terzi C.
      • Bingul M.
      • Arslan N.C.
      • Ozturk E.
      • Canda A.E.
      • Isik O.
      • et al.
      Randomized controlled trial of 8 weeks' vs 12 weeks' interval between neoadjuvant chemoradiotherapy and surgery for locally advanced rectal cancer.
      ] including a total number of 657 patients, comparing classic intervals (between 6 and 8 weeks) with prolonged intervals (10 weeks and 12 weeks respectively) there was an increase in pathological complete response and downstaging, but not in TRG.

      Tumour downstaging

      Downstaging is based on the clinical stage of the tumour before the start of the neoadjuvant treatment (cTNM) in comparison with post-treatment stage, either on imaging or endoscopic evaluation (ycTNM) or in resection specimens (ypTNM). In principle, this should be straightforward, either there is tumour present in a certain layer of the bowel wall or it is not present. This relatively elegant approach has, however, a number of disadvantages. Clinical staging is highly variable since the current imaging modalities are not always accurate and/or endoscopic experience is sometimes inadequate, given the large differences between clinical and pathological stage [

      Brouwer NPM, Stijns RCH, Lemmens V, Nagtegaal ID, Beets-Tan RGH, Futterer JJ, et al. Clinical lymph node staging in colorectal cancer; a flip of the coin? Europ J Surg Oncol: J Europ Soc Surg Oncol Brit Assoc Surg Oncol 2018;44:1241-6.

      ]. Even in the most simplified version of downstaging, focusing on complete response (i.e. no tumour present after neoadjuvant therapy), differences between clinical staging and pathological staging are profound [
      • Uzcudun A.E.
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      Efficacy of preoperative radiation therapy for resectable rectal adenocarcinoma when combined with oral tegafur-uracil modulated with leucovorin: results from a phase II study.
      ,
      • Burke S.J.
      • Percarpio B.A.
      • Knight D.C.
      • Kwasnik E.M.
      Combined preoperative radiation and mitomycin/5-fluorouracil treatment for locally advanced rectal adenocarcinoma.
      ,
      • Benzoni E.
      • Cerato F.
      • Cojutti A.
      • Milan E.
      • Pontello D.
      • Chiaulon G.
      • et al.
      The predictive value of clinical evaluation of response to neoadjuvant chemoradiation therapy for rectal cancer.
      ,

      Bujko K, Richter P, Kolodziejczyk M, Nowacki MP, Kulig J, Popiela T, et al. Preoperative radiotherapy and local excision of rectal cancer with immediate radical re-operation for poor responders. Radiother Oncol: J Europ Soc Therap Radiol Oncol 2009;92:195–201.

      ,
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      • Chang C.C.
      • Kung C.H.
      • Lin S.E.
      • et al.
      Can we predict pathologic complete response before surgery for locally advanced rectal cancer treated with preoperative chemoradiation therapy?.
      ,
      • Guillem J.G.
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      • Shia J.
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      • Mazumdar M.
      • Bernard B.
      • et al.
      Clinical examination following preoperative chemoradiation for rectal cancer is not a reliable surrogate end point.
      ,
      • Meterissian S.
      • Skibber J.
      • Rich T.
      • Roubein L.
      • Ajani J.
      • Cleary K.
      • et al.
      Patterns of residual disease after preoperative chemoradiation in ultrasound T3 rectal carcinoma.
      ,
      • Lopez-Lopez V.
      • Abrisqueta J.
      • Lujan J.
      • Hernandez Q.
      • Ono A.
      • Parrilla P.
      Utility of rectoscopy in the assessment of response to neoadjuvant treatment for locally advanced rectal cancer.
      ,
      • Lezoche E.
      • Guerrieri M.
      • Paganini A.M.
      • Baldarelli M.
      • De Sanctis A.
      • Lezoche G.
      Long-term results in patients with T2–3 N0 distal rectal cancer undergoing radiotherapy before transanal endoscopic microsurgery.
      ,
      • Kim C.J.
      • Yeatman T.J.
      • Coppola D.
      • Trotti A.
      • Williams B.
      • Barthel J.S.
      • et al.
      Local excision of T2 and T3 rectal cancers after downstaging chemoradiation.
      ,
      • Smith F.M.
      • Wiland H.
      • Mace A.
      • Pai R.K.
      • Kalady M.F.
      Depth and lateral spread of microscopic residual rectal cancer after neoadjuvant chemoradiation: implications for treatment decisions.
      ,

      Chari RS, Tyler DS, Anscher MS, Russell L, Clary BM, Hathorn J, et al. Preoperative radiation and chemotherapy in the treatment of adenocarcinoma of the rectum. Ann Surgery 1995;221:778–86; discussion 86-7.

      ,
      • Tulchinsky H.
      • Rabau M.
      • Shacham-Shemueli E.
      • Goldman G.
      • Geva R.
      • Inbar M.
      • et al.
      Can rectal cancers with pathologic T0 after neoadjuvant chemoradiation (ypT0) be treated by transanal excision alone?.
      ,
      • Zmora O.
      • Dasilva G.M.
      • Gurland B.
      • Pfeffer R.
      • Koller M.
      • Nogueras J.J.
      • et al.
      Does rectal wall tumor eradication with preoperative chemoradiation permit a change in the operative strategy?.
      ,

      Hiotis SP, Weber SM, Cohen AM, Minsky BD, Paty PB, Guillem JG, et al. Assessing the predictive value of clinical complete response to neoadjuvant therapy for rectal cancer: an analysis of 488 patients. J Am College Surgeons 2002;194:131–5; discussion 5-6.

      ,
      • Bonnen M.
      • Crane C.
      • Vauthey J.N.
      • Skibber J.
      • Delclos M.E.
      • Rodriguez-Bigas M.
      • et al.
      Long-term results using local excision after preoperative chemoradiation among selected T3 rectal cancer patients.
      ,
      • Smith F.M.
      • Chang K.H.
      • Sheahan K.
      • Hyland J.
      • O'Connell P.R.
      • Winter D.C.
      The surgical significance of residual mucosal abnormalities in rectal cancer following neoadjuvant chemoradiotherapy.
      ,
      • Koh D.M.
      • Chau I.
      • Tait D.
      • Wotherspoon A.
      • Cunningham D.
      • Brown G.
      Evaluating mesorectal lymph nodes in rectal cancer before and after neoadjuvant chemoradiation using thin-section T2-weighted magnetic resonance imaging.
      ,
      • Onaitis M.W.
      • Noone R.B.
      • Fields R.
      • Hurwitz H.
      • Morse M.
      • Jowell P.
      • et al.
      Complete response to neoadjuvant chemoradiation for rectal cancer does not influence survival.
      ] (Fig. 1). Of the 576 cases of clinical complete response described in the literature, only 279 presented with a complete pathological response after surgery (45%) [
      • Uzcudun A.E.
      • Batlle J.F.
      • Velasco J.C.
      • Sanchez Santos M.E.
      • Carpeno Jde C.
      • Grande A.G.
      • et al.
      Efficacy of preoperative radiation therapy for resectable rectal adenocarcinoma when combined with oral tegafur-uracil modulated with leucovorin: results from a phase II study.
      ,
      • Burke S.J.
      • Percarpio B.A.
      • Knight D.C.
      • Kwasnik E.M.
      Combined preoperative radiation and mitomycin/5-fluorouracil treatment for locally advanced rectal adenocarcinoma.
      ,
      • Benzoni E.
      • Cerato F.
      • Cojutti A.
      • Milan E.
      • Pontello D.
      • Chiaulon G.
      • et al.
      The predictive value of clinical evaluation of response to neoadjuvant chemoradiation therapy for rectal cancer.
      ,

      Bujko K, Richter P, Kolodziejczyk M, Nowacki MP, Kulig J, Popiela T, et al. Preoperative radiotherapy and local excision of rectal cancer with immediate radical re-operation for poor responders. Radiother Oncol: J Europ Soc Therap Radiol Oncol 2009;92:195–201.

      ,
      • Kuo L.J.
      • Chiou J.F.
      • Tai C.J.
      • Chang C.C.
      • Kung C.H.
      • Lin S.E.
      • et al.
      Can we predict pathologic complete response before surgery for locally advanced rectal cancer treated with preoperative chemoradiation therapy?.
      ,
      • Guillem J.G.
      • Chessin D.B.
      • Shia J.
      • Moore H.G.
      • Mazumdar M.
      • Bernard B.
      • et al.
      Clinical examination following preoperative chemoradiation for rectal cancer is not a reliable surrogate end point.
      ,
      • Meterissian S.
      • Skibber J.
      • Rich T.
      • Roubein L.
      • Ajani J.
      • Cleary K.
      • et al.
      Patterns of residual disease after preoperative chemoradiation in ultrasound T3 rectal carcinoma.
      ,
      • Lopez-Lopez V.
      • Abrisqueta J.
      • Lujan J.
      • Hernandez Q.
      • Ono A.
      • Parrilla P.
      Utility of rectoscopy in the assessment of response to neoadjuvant treatment for locally advanced rectal cancer.
      ,
      • Lezoche E.
      • Guerrieri M.
      • Paganini A.M.
      • Baldarelli M.
      • De Sanctis A.
      • Lezoche G.
      Long-term results in patients with T2–3 N0 distal rectal cancer undergoing radiotherapy before transanal endoscopic microsurgery.
      ,
      • Kim C.J.
      • Yeatman T.J.
      • Coppola D.
      • Trotti A.
      • Williams B.
      • Barthel J.S.
      • et al.
      Local excision of T2 and T3 rectal cancers after downstaging chemoradiation.
      ,
      • Smith F.M.
      • Wiland H.
      • Mace A.
      • Pai R.K.
      • Kalady M.F.
      Depth and lateral spread of microscopic residual rectal cancer after neoadjuvant chemoradiation: implications for treatment decisions.
      ,

      Chari RS, Tyler DS, Anscher MS, Russell L, Clary BM, Hathorn J, et al. Preoperative radiation and chemotherapy in the treatment of adenocarcinoma of the rectum. Ann Surgery 1995;221:778–86; discussion 86-7.

      ,
      • Tulchinsky H.
      • Rabau M.
      • Shacham-Shemueli E.
      • Goldman G.
      • Geva R.
      • Inbar M.
      • et al.
      Can rectal cancers with pathologic T0 after neoadjuvant chemoradiation (ypT0) be treated by transanal excision alone?.
      ,
      • Zmora O.
      • Dasilva G.M.
      • Gurland B.
      • Pfeffer R.
      • Koller M.
      • Nogueras J.J.
      • et al.
      Does rectal wall tumor eradication with preoperative chemoradiation permit a change in the operative strategy?.
      ,

      Hiotis SP, Weber SM, Cohen AM, Minsky BD, Paty PB, Guillem JG, et al. Assessing the predictive value of clinical complete response to neoadjuvant therapy for rectal cancer: an analysis of 488 patients. J Am College Surgeons 2002;194:131–5; discussion 5-6.

      ,
      • Bonnen M.
      • Crane C.
      • Vauthey J.N.
      • Skibber J.
      • Delclos M.E.
      • Rodriguez-Bigas M.
      • et al.
      Long-term results using local excision after preoperative chemoradiation among selected T3 rectal cancer patients.
      ,
      • Smith F.M.
      • Chang K.H.
      • Sheahan K.
      • Hyland J.
      • O'Connell P.R.
      • Winter D.C.
      The surgical significance of residual mucosal abnormalities in rectal cancer following neoadjuvant chemoradiotherapy.
      ,
      • Koh D.M.
      • Chau I.
      • Tait D.
      • Wotherspoon A.
      • Cunningham D.
      • Brown G.
      Evaluating mesorectal lymph nodes in rectal cancer before and after neoadjuvant chemoradiation using thin-section T2-weighted magnetic resonance imaging.
      ,
      • Onaitis M.W.
      • Noone R.B.
      • Fields R.
      • Hurwitz H.
      • Morse M.
      • Jowell P.
      • et al.
      Complete response to neoadjuvant chemoradiation for rectal cancer does not influence survival.
      ]. Considerable differences were present in the workup of these different cohorts, and large studies with standardised evaluation are lacking.
      Figure thumbnail gr1
      Fig. 1Comparison between clinical complete response and pathological complete response (primary tumour only) in cases with resection after neoadjuvant chemoradiation [
      • Uzcudun A.E.
      • Batlle J.F.
      • Velasco J.C.
      • Sanchez Santos M.E.
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      Bujko K, Richter P, Kolodziejczyk M, Nowacki MP, Kulig J, Popiela T, et al. Preoperative radiotherapy and local excision of rectal cancer with immediate radical re-operation for poor responders. Radiother Oncol: J Europ Soc Therap Radiol Oncol 2009;92:195–201.

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      Depth and lateral spread of microscopic residual rectal cancer after neoadjuvant chemoradiation: implications for treatment decisions.
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      Chari RS, Tyler DS, Anscher MS, Russell L, Clary BM, Hathorn J, et al. Preoperative radiation and chemotherapy in the treatment of adenocarcinoma of the rectum. Ann Surgery 1995;221:778–86; discussion 86-7.

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      Hiotis SP, Weber SM, Cohen AM, Minsky BD, Paty PB, Guillem JG, et al. Assessing the predictive value of clinical complete response to neoadjuvant therapy for rectal cancer: an analysis of 488 patients. J Am College Surgeons 2002;194:131–5; discussion 5-6.

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      • Bonnen M.
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      • Skibber J.
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      Long-term results using local excision after preoperative chemoradiation among selected T3 rectal cancer patients.
      ,
      • Smith F.M.
      • Chang K.H.
      • Sheahan K.
      • Hyland J.
      • O'Connell P.R.
      • Winter D.C.
      The surgical significance of residual mucosal abnormalities in rectal cancer following neoadjuvant chemoradiotherapy.
      ,
      • Koh D.M.
      • Chau I.
      • Tait D.
      • Wotherspoon A.
      • Cunningham D.
      • Brown G.
      Evaluating mesorectal lymph nodes in rectal cancer before and after neoadjuvant chemoradiation using thin-section T2-weighted magnetic resonance imaging.
      ,
      • Onaitis M.W.
      • Noone R.B.
      • Fields R.
      • Hurwitz H.
      • Morse M.
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      Complete response to neoadjuvant chemoradiation for rectal cancer does not influence survival.
      ]. The bars represent the total number of cases with a clinical complete response, the green part of the bar represent the cases where the clinical complete response is confirmed by a pathological complete response. For each study, the methods of determination of the clinical complete response are given: D: digital rectal examination, E: endoscopy (with or without biopsies), C: CT, M: MRI, U: ultrasound, *: not described. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
      One of the problems of downstaging is the lack of integration with response patterns: in reaction to treatment there is profound heterogeneity even within one tumour. This causes an important difference between staging in tumours with or without neoadjuvant treatment: after neoadjuvant treatment, tumour can remain present in the outer layers of the bowel wall, while disappearing in the inner layers, as has been shown in two independent studies [
      • Duldulao M.P.
      • Lee W.
      • Streja L.
      • Chu P.
      • Li W.
      • Chen Z.
      • et al.
      Distribution of residual cancer cells in the bowel wall after neoadjuvant chemoradiation in patients with rectal cancer.
      ,
      • Xiao L.
      • Yu X.
      • Deng W.
      • Feng H.
      • Chang H.
      • Xiao W.
      • et al.
      Pathological assessment of rectal cancer after neoadjuvant chemoradiotherapy: distribution of residual cancer cells and accuracy of biopsy.
      ] (summarized in Fig. 2). This phenomenon seriously threatens the oncological safety local excisions after neoadjuvant treatment.
      Figure thumbnail gr2
      Fig. 2The distribution of residual tumour cells within the rectal wall, including 7 patients with ypTis (patient 1–7), 16 patients with ypT1, 79 patients with ypT2, 127 patients with ypT3 and 8 patients with ypT4 rectal cancer. Along the x-axis the individual patients are depicted. Submucosal tumour was absent in 116 patients with ypT2-4 cancer (patient number 121–237, 54%). Adapted and combined data from Duldulao et al.
      [
      • Duldulao M.P.
      • Lee W.
      • Streja L.
      • Chu P.
      • Li W.
      • Chen Z.
      • et al.
      Distribution of residual cancer cells in the bowel wall after neoadjuvant chemoradiation in patients with rectal cancer.
      ]
      and Xiao et al.
      [
      • Xiao L.
      • Yu X.
      • Deng W.
      • Feng H.
      • Chang H.
      • Xiao W.
      • et al.
      Pathological assessment of rectal cancer after neoadjuvant chemoradiotherapy: distribution of residual cancer cells and accuracy of biopsy.
      ]
      .
      Invasion depth is only one of the features of staging, the second issue is nodal status. In the 'surgery only' situation, there is a clear relation between invasion depth and nodal stage. Lymph node risk is dependent on several features reflecting tumour biology, including differentiation grade, interaction with the microenvironment (i.e. tumour budding), invasion of lymphatic vessels and histological type [
      • Bosch S.L.
      • Teerenstra S.
      • de Wilt J.H.
      • Cunningham C.
      • Nagtegaal I.D.
      Predicting lymph node metastasis in pT1 colorectal cancer: a systematic review of risk factors providing rationale for therapy decisions.
      ]. Not all patients with complete tumour regression after neoadjuvant therapy also have complete regression of lymph node metastases. Remaining tumour in lymph nodes after a complete response of the primary tumour is estimated at 7% [
      • Nagtegaal I.D.
      • Marijnen C.A.M.
      The future of TNM staging in rectal cancer; the era of neoadjuvant therapy.
      ], demonstrating again the heterogeneity of therapy response. When the rests of the primary tumour are restricted to the submucosa (ypT1), the lymph node risk is comparable to the risk in non-neoadjuvant treated tumours in the submucosa (11%) [
      • Bosch S.L.
      • Teerenstra S.
      • de Wilt J.H.
      • Cunningham C.
      • Nagtegaal I.D.
      Predicting lymph node metastasis in pT1 colorectal cancer: a systematic review of risk factors providing rationale for therapy decisions.
      ]. Moreover, in the neoadjuvant setting it no longer easy to predict lymph node status based on histological features. Only tumour grading in the post-treatment specimen, in combination with remaining tumour diameter and clinical assessment of the nodal status showed some predictive value [
      • Bosch S.L.
      • Vermeer T.A.
      • West N.P.
      • Swellengrebel H.A.
      • Marijnen C.A.
      • Cats A.
      • et al.
      Clinicopathological characteristics predict lymph node metastases in ypT0-2 rectal cancer after chemoradiotherapy.
      ].
      It is thus important to realize that the impact of TNM stage after neoadjuvant treatment (ypTNM) is no longer identical to stage in the 'surgery only' setting (pTNM) [
      • Bosch S.L.
      • Verhoeven R.H.A.
      • Lemmens V.
      • Simmer F.
      • Poortmans P.
      • de Wilt J.H.W.
      • et al.
      Type of preoperative therapy and stage-specific survival after surgery for rectal cancer: a nationwide population-based cohort study.
      ]. In stage I patients (either early tumours or good responders), outcomes are good in both settings. However the prognosis of both ypTNM II and III (intermediate responders) is significantly worse than that of pTNM II and III. This is not unexpected, since these cases present with only a limited (if any) response to the neoadjuvant therapy and thus form a subselection of tumours with a poor outcome.

      Tumour regression grading systems

      Since the tumour response does not always result in downstaging, a more morphological approach was adapted: tumour regression grading (TRG). The basis of most TRG systems is the Mandard classification [
      • Mandard A.M.
      • Dalibard F.
      • Mandard J.C.
      • Marnay J.
      • Henry-Amar M.
      • Petiot J.F.
      • et al.
      Pathologic assessment of tumor regression after preoperative chemoradiotherapy of esophageal carcinoma. Clinicopathologic correlations.
      ], that was described in a series of squamous cell carcinomas of the oesophagus, without any clinical impact. Translation into TRG for rectal cancer was described in 17 patients by Dworak [
      • Dworak O.
      • Keilholz L.
      • Hoffmann A.
      Pathological features of rectal cancer after preoperative radiochemotherapy.
      ], who slightly changed the definitions that were used for the grades between complete response and no response. The initial TRG systems were 5-tiered, subsequent developments resulted in 3- and 4-tiered systems, most with subtle differences from the original classification. A radical different approach was proposed by Rodel [
      • Rodel C.
      • Martus P.
      • Papadoupolos T.
      • Fuzesi L.
      • Klimpfinger M.
      • Fietkau R.
      • et al.
      Prognostic significance of tumor regression after preoperative chemoradiotherapy for rectal cancer.
      ], and used the percentage of response. Similarly, the percentage of residual cells is estimated in the Aberdeen/Murray scale [
      • Brown G.T.
      • Cash B.
      • Alnabulsi A.
      • Samuel L.M.
      • Murray G.I.
      The expression and prognostic significance of bcl-2-associated transcription factor 1 in rectal cancer following neoadjuvant therapy.
      ]. While these are valid approaches in imaging studies, since before and after situations can easily be compared, this method is not applicable to the histologic TRG. To complicate matters even further, TRG0 does refer either to cases with no response [
      • Dworak O.
      • Keilholz L.
      • Hoffmann A.
      Pathological features of rectal cancer after preoperative radiochemotherapy.
      ,
      • Rodel C.
      • Martus P.
      • Papadoupolos T.
      • Fuzesi L.
      • Klimpfinger M.
      • Fietkau R.
      • et al.
      Prognostic significance of tumor regression after preoperative chemoradiotherapy for rectal cancer.
      ] or with no residual tumour cells or complete response [

      AJCC Cancer Staging Manual. Chicago: Springer; 2017.

      ].
      Most published papers demonstrate a relation with TRG and patient outcome. However, most 5-tiered TRG studies end up with a simplified version (3-tiered) to provide us with significant results. The large variation in definitions and classifications hamper the implementation of TRG in clinical practice. National guidelines therefore advocate simplified versions, along the lines of regression: yes/no/partly [
      • Glynne-Jones R.
      • Wyrwicz L.
      • Tiret E.
      • Brown G.
      • Rodel C.
      • Cervantes A.
      • et al.
      Rectal cancer: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up.
      ], rather than using the more detailed systems described in the literature. Moreover, there is a large variation in the preference of pathologists for specific TRG versions to be used in daily practice, that cannot be explained by national or international guidelines [

      Westerhoff M, Osecky M, Langer R. Varying practices in tumor regression grading of gastrointestinal carcinomas after neoadjuvant therapy - results of an international survey. Mod Pathol: Off J United States Can Acad Pathol, Inc. 2019;32:141.

      ].
      Several studies have evaluated the interobserver variability of TRG and inevitably demonstrate little overall agreement [
      • Chetty R.
      • Gill P.
      • Govender D.
      • Bateman A.
      • Chang H.J.
      • Deshpande V.
      • et al.
      International study group on rectal cancer regression grading: interobserver variability with commonly used regression grading systems.
      ]. Moreover, when pathologists are blinded for treatment arm, up to 32% of cases that have not received any neoadjuvant treatment, are scored as showing at least some tumour regression [
      • Vironen J.
      • Juhola M.
      • Kairaluoma M.
      • Jantunen I.
      • Kellokumpu I.
      Tumour regression grading in the evaluation of tumour response after different preoperative radiotherapy treatments for rectal carcinoma.
      ]. It has been suggested that diligence of the pathologist is also an important factor. However, a recent study [
      • Chow O.S.
      • Patil S.
      • Keskin M.
      • Smith J.J.
      • Widmar M.
      • Smith D.D.
      • et al.
      Variation in the thoroughness of pathologic assessment and response rates of locally advanced rectal cancers after chemoradiation.
      ] examining thoroughness of pathologic assessment could not demonstrate a difference, when evaluating the number of examined tissue blocks. In conclusion, TRG is not specific, not reproducible and there is no consensus about its use. Different scales measure different things. However, when cases of rectal cancer are compared after neoadjuvant treatment, striking differences in histology and outcome of patients are observed (Fig. 3). Therefore, it seems that we are missing the point.
      Figure thumbnail gr3
      Fig. 3Histological illustrations of tumour response. A. Absence of response, adenocarcinoma with little stromal reaction and a high tumour cell percentage. B. Extensive tumour response, with increased stroma between tumour glands. C. Mucin pool formation in the deeper areas of the tumour, the mucin pools are acellular. HE stain.

      Patterns of tumour response: Fragmentation versus shrinkage

      The simplified idea that when a tumour responds to treatment it shrinks towards the lumen of the bowel is the basis of either local excision or watchful waiting after neoadjuvant therapy. This would be very convenient, and is possible if the tumour has not breached the muscularis propria, allowing for biopsy-guided treatment decisions. However, with locally advanced tumours that have grown into the deeper layers of the mesorectal fatty tissue, or even beyond, in prostate or vagina, this concept is more fanciful and not reliable. We postulated in 2007 [
      • Gosens M.J.
      • Klaassen R.A.
      • Tan-Go I.
      • Rutten H.J.
      • Martijn H.
      • van den Brule A.J.
      • et al.
      Circumferential margin involvement is the crucial prognostic factor after multimodality treatment in patients with locally advanced rectal carcinoma.
      ] that there are at least two ways in which rectal cancer can respond to neoadjuvant treatment (Fig. 4): shrinkage or fragmentation. Shrinkage (in the direction of the mucosa) is of course the preferred mode of response, allowing adequate monitoring by imaging or endoscopy and perhaps limited treatment. This is also the rationale for watch and wait strategies. Fragmentation, i.e. the destruction of the main tumour mass and formation of small groups of tumour cells, is more difficult to deal with. The limited size of the fragments is below the power of resolution on imaging and surgical treatment might be irradical, resulting in local recurrences. Three recent studies (289 patients with locally advanced rectal cancer) [
      • Perez R.O.
      • Habr-Gama A.
      • Smith F.M.
      • Kosinski L.
      • Sao Juliao G.P.
      • Grzona E.
      • et al.
      Fragmented pattern of tumor regression and lateral intramural spread may influence margin appropriateness after TEM for rectal cancer following neoadjuvant CRT.
      ,
      • Hav M.
      • Libbrecht L.
      • Geboes K.
      • Ferdinande L.
      • Boterberg T.
      • Ceelen W.
      • et al.
      Prognostic value of tumor shrinkage versus fragmentation following radiochemotherapy and surgery for rectal cancer.
      ,
      • Fernandez-Acenero M.J.
      • Estrada Munoz L.
      • Sastre Varela J.
      • Corona Sanchez J.A.
      • Diaz Del Arco C.
      • Garcia Paredes B.
      • et al.
      Prognostic influence of histopathological regression patterns in rectal adenocarcinoma receiving neoadjuvant therapy.
      ] showed that fragmentation was present in 40% of cases, while in a study of 45 cT3 patients from Cleveland [
      • Smith F.M.
      • Wiland H.
      • Mace A.
      • Pai R.K.
      • Kalady M.F.
      Depth and lateral spread of microscopic residual rectal cancer after neoadjuvant chemoradiation: implications for treatment decisions.
      ] 80% of cases showed fragmented response. Fragmentation or scattering of tumour cells also causes a clear discrepancy between macroscopy and microscopy [
      • Hayden D.M.
      • Jakate S.
      • Pinzon M.C.
      • Giusto D.
      • Francescatti A.B.
      • Brand M.I.
      • et al.
      Tumor scatter after neoadjuvant therapy for rectal cancer: are we dealing with an invisible margin?.
      ]. In a careful description of the findings in 27 patients (49%), it was shown that fragments could be found up to 3 cm from the ulcer, in all directions (proximal, distal and lateral). Fragmentation is associated with more frequent residual lymph node metastases, less downstaging, positive resection margins and a poor outcome [
      • Perez R.O.
      • Habr-Gama A.
      • Smith F.M.
      • Kosinski L.
      • Sao Juliao G.P.
      • Grzona E.
      • et al.
      Fragmented pattern of tumor regression and lateral intramural spread may influence margin appropriateness after TEM for rectal cancer following neoadjuvant CRT.
      ,
      • Hav M.
      • Libbrecht L.
      • Geboes K.
      • Ferdinande L.
      • Boterberg T.
      • Ceelen W.
      • et al.
      Prognostic value of tumor shrinkage versus fragmentation following radiochemotherapy and surgery for rectal cancer.
      ,
      • Fernandez-Acenero M.J.
      • Estrada Munoz L.
      • Sastre Varela J.
      • Corona Sanchez J.A.
      • Diaz Del Arco C.
      • Garcia Paredes B.
      • et al.
      Prognostic influence of histopathological regression patterns in rectal adenocarcinoma receiving neoadjuvant therapy.
      ]. When TRG was applied to these cases [
      • Hav M.
      • Libbrecht L.
      • Geboes K.
      • Ferdinande L.
      • Boterberg T.
      • Ceelen W.
      • et al.
      Prognostic value of tumor shrinkage versus fragmentation following radiochemotherapy and surgery for rectal cancer.
      ], there was no prognostic impact. However, one could argue that these were cases with intermediate TRG grades.
      Figure thumbnail gr4
      Fig. 4Schematic representation of response patterns. A. Pretreatment situation: the tumour is locally advanced (in black) and breached the circumferential resection margin. This pretreatment contour is also depicted in B, C, D. B. “Tumour shrinkage” scenario in which there is a decreased invasion depth (downstaging, but not necessarily a better TRG), C. Mucin pool formation (blue), due to lack of tumour cells in the mucin lakes, these are not taken into account for staging (downstaging, variable effect on TRG) D. “Tumour fragmentation” scenario in which scattered groups of tumour cells spread throughout the whole fibrotic area, retaining the original infiltration depth (no downstaging, but better TRG). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

       The third form of response: mucin pool formation

      The relation between mucin pool formation, also described as colloid response, and neoadjuvant therapy has been known for a long time. Even short-term neoadjuvant radiotherapy regimens result in a significant increase of tumours classified as mucinous carcinomas [
      • Nagtegaal I.D.
      • Gaspar C.S.G.
      • Marijnen C.A.M.
      • Mulder A.
      • van de Velde C.J.H.
      • Fodde R.
      • et al.
      Morphological changes in tumour type after radiotherapy are accompanied by changes in gene expression profile but not in clinical behaviour.
      ]. Longer after treatment, not all mucin pools remain cellular, and the typical acellular mucin pools (Fig. 3c) present as part of therapy response. In patients with a complete pathological response, 27% of patients present with acellular mucin pools [
      • Bhatti A.B.H.
      • Zaheer S.
      • Shafique K.
      Prognostic role of acellular mucin pools in patients with rectal cancer after pathological complete response to preoperative chemoradiation: systematic review and meta-analysis.
      ], while in all stages this was between 12 and 35% [
      • Fernandez-Acenero M.J.
      • Estrada Munoz L.
      • Sastre Varela J.
      • Corona Sanchez J.A.
      • Diaz Del Arco C.
      • Garcia Paredes B.
      • et al.
      Prognostic influence of histopathological regression patterns in rectal adenocarcinoma receiving neoadjuvant therapy.
      ,

      Cienfuegos JA, Baixauli J, Rotellar F, Arredondo J, Sola JJ, Arbea L, et al. Clinical significance of cellular and acellular mucin pools in rectal carcinoma following preoperative chemoradiotherapy. Clin Trans Oncol: Off Publ Federation Spanish Oncol Soc Nat Cancer Inst Mexico 2016;18:714–21.

      ,
      • Reynolds I.S.
      • McNamara D.A.
      • Kay E.W.
      • O'Neill B.
      • Deasy J.
      • Burke J.P.
      The significance of mucin pools following neoadjuvant chemoradiotherapy for locally advanced rectal cancer.
      ,
      • Kang C.M.
      • Lim S.B.
      • Hong S.M.
      • Yu C.S.
      • Hong Y.S.
      • Kim T.W.
      • et al.
      Prevalence and clinical significance of cellular and acellular mucin in patients with locally advanced mucinous rectal cancer who underwent preoperative chemoradiotherapy followed by radical surgery.
      ,
      • Swellengrebel H.A.
      • Bosch S.L.
      • Cats A.
      • Vincent A.D.
      • Dewit L.G.
      • Verwaal V.J.
      • et al.
      Tumour regression grading after chemoradiotherapy for locally advanced rectal cancer: a near pathologic complete response does not translate into good clinical outcome.
      ]. The presence of acellular mucin pools in pCR patients does not affect prognosis [
      • Bhatti A.B.H.
      • Zaheer S.
      • Shafique K.
      Prognostic role of acellular mucin pools in patients with rectal cancer after pathological complete response to preoperative chemoradiation: systematic review and meta-analysis.
      ], while some studies suggest better outcome for all stages in the presence of mucin pools [
      • Fernandez-Acenero M.J.
      • Estrada Munoz L.
      • Sastre Varela J.
      • Corona Sanchez J.A.
      • Diaz Del Arco C.
      • Garcia Paredes B.
      • et al.
      Prognostic influence of histopathological regression patterns in rectal adenocarcinoma receiving neoadjuvant therapy.
      ,

      Cienfuegos JA, Baixauli J, Rotellar F, Arredondo J, Sola JJ, Arbea L, et al. Clinical significance of cellular and acellular mucin pools in rectal carcinoma following preoperative chemoradiotherapy. Clin Trans Oncol: Off Publ Federation Spanish Oncol Soc Nat Cancer Inst Mexico 2016;18:714–21.

      ,
      • Reynolds I.S.
      • McNamara D.A.
      • Kay E.W.
      • O'Neill B.
      • Deasy J.
      • Burke J.P.
      The significance of mucin pools following neoadjuvant chemoradiotherapy for locally advanced rectal cancer.
      ]. In contrast, presence of remaining tumour cells in mucin pools is associated with poor outcome [

      Cienfuegos JA, Baixauli J, Rotellar F, Arredondo J, Sola JJ, Arbea L, et al. Clinical significance of cellular and acellular mucin pools in rectal carcinoma following preoperative chemoradiotherapy. Clin Trans Oncol: Off Publ Federation Spanish Oncol Soc Nat Cancer Inst Mexico 2016;18:714–21.

      ]. Some studies do not distinguish between cellular and acellular mucin pools, showing intermediate outcomes in this group [
      • Rullier A.
      • Laurent C.
      • Vendrely V.
      • Le B.B.
      • Bioulac-Sage P.
      • Rullier E.
      Impact of colloid response on survival after preoperative radiotherapy in locally advanced rectal carcinoma.
      ].
      The exact mechanism of colloid response or mucinous degeneration is not clear. This kind of reaction is observed more frequent relative early after treatment in pCR patients [
      • Lim S.B.
      • Hong S.M.
      • Yu C.S.
      • Hong Y.S.
      • Kim T.W.
      • Park J.H.
      • et al.
      Prevalence and clinical significance of acellular mucin in locally advanced rectal cancer patients showing pathologic complete response to preoperative chemoradiotherapy.
      ], but also after chemotherapy [
      • Suarez J.
      • Amat I.
      • Vera R.
      • Balen E.
      • Gomez M.
      • Lera J.M.
      Pathologic response of primary rectal cancer to oxaliplatin-based chemotherapy.
      ] and in cases of epithelial misplacement (pseudoinvasion) in early colorectal cancer [
      • Panarelli N.C.
      • Somarathna T.
      • Samowitz W.S.
      • Kornacki S.
      • Sanders S.A.
      • Novelli M.R.
      • et al.
      Diagnostic challenges caused by endoscopic biopsy of colonic polyps: a systematic evaluation of epithelial misplacement with review of problematic polyps from the bowel cancer screening program, United Kingdom.
      ], suggesting that this is more general response mechanism. Mucoid degeneration is, for example, also observed in orthopaedics, in the anterior cruciate ligament of the knee [
      • Pandey V.
      • Suman C.
      • Sharma S.
      • Rao S.P.
      • Kiran Acharya K.
      • Sambaji C.
      Mucoid degeneration of the anterior cruciate ligament: management and outcome.
      ]. Theories about the aetiology of this phenomenon include repeated microtrauma, where fibroblasts excrete glycosaminoglycans or hyaluronic acid, degeneration as an (accelerated) aging process and proliferation of pluripotent mesenchymal stem cells. Whether any of these processes is also involved in post-treatment mucin pool formation is unknown.

      Clinical implications of the different patterns of tumour response

      If fragmentation is indeed a response mechanism that is present in a relevant number of cases, then this should be considered in clinical care since there might be residual tumour cells present in the deeper layers of the bowel wall. This would then explain:
      • 1.
        Differences between downstaging and TRG
      • 2.
        Higher local regrowth in advanced tumours during watchful waiting procedures
      • 3.
        The importance of positive margins over TRG and downstaging
      • 4.
        The limited value of post-treatment biopsies
      • 5.
        The poor outcome of patients with near complete pCR
      • 6.
        The biology of tumour deposits after neoadjuvant therapy

       Differences between downstaging and TRG

      Several studies have analysed both downstaging and TRG [
      • Bouzourene H.
      • Bosman F.T.
      • Seelentag W.
      • Matter M.
      • Coucke P.
      Importance of tumor regression assessment in predicting the outcome in patients with locally advanced rectal carcinoma who are treated with preoperative radiotherapy.
      ,
      • Park Y.J.
      • Oh B.R.
      • Lim S.W.
      • Huh J.W.
      • Joo J.K.
      • Kim Y.J.
      • et al.
      Clinical significance of tumor regression grade in rectal cancer with preoperative chemoradiotherapy.
      ,
      • Kim N.K.
      • Baik S.H.
      • Min B.S.
      • Pyo H.R.
      • Choi Y.J.
      • Kim H.
      • et al.
      A comparative study of volumetric analysis, histopathologic downstaging, and tumor regression grade in evaluating tumor response in locally advanced rectal cancer following preoperative chemoradiation.
      ,
      • Xu L.
      • Cai S.
      • Xiao T.
      • Chen Y.
      • Qiu H.
      • Wu B.
      • et al.
      Prognostic significance of tumour regression grade after neoadjuvant chemoradiotherapy for a cohort of patients with locally advanced rectal cancer: an 8-year retrospective single-institutional study.
      ,
      • Reggiani Bonetti L.
      • Lionti S.
      • Domati F.
      • Barresi V.
      Do pathological variables have prognostic significance in rectal adenocarcinoma treated with neoadjuvant chemoradiotherapy and surgery?.
      ,
      • Rullier A.
      • Laurent C.
      • Capdepont M.
      • Vendrely V.
      • Bioulac-Sage P.
      • Rullier E.
      Impact of tumor response on survival after radiochemotherapy in locally advanced rectal carcinoma.
      ] (Fig. 5), showing that tumour regression is not always accompanied by downstaging. This difference can be explained by the pattern of response: downstaging is accompanying TRG only in cases of tumour shrinkage, while fragmentation does not lead to T downstaging. In addition, the prognostic value of tumour response in these studies differs, with independent prognostic value of either downstaging [
      • Rullier A.
      • Laurent C.
      • Capdepont M.
      • Vendrely V.
      • Bioulac-Sage P.
      • Rullier E.
      Impact of tumor response on survival after radiochemotherapy in locally advanced rectal carcinoma.
      ] or TRG [
      • Xu L.
      • Cai S.
      • Xiao T.
      • Chen Y.
      • Qiu H.
      • Wu B.
      • et al.
      Prognostic significance of tumour regression grade after neoadjuvant chemoradiotherapy for a cohort of patients with locally advanced rectal cancer: an 8-year retrospective single-institutional study.
      ] for disease-free and overall survival.
      Figure thumbnail gr5
      Fig. 5Correlation between downstaging and tumour regression grading. The size of the bubble indicates the size of the cohort. In total 933 patients from 6 studies are included [
      • Bouzourene H.
      • Bosman F.T.
      • Seelentag W.
      • Matter M.
      • Coucke P.
      Importance of tumor regression assessment in predicting the outcome in patients with locally advanced rectal carcinoma who are treated with preoperative radiotherapy.
      ,
      • Park Y.J.
      • Oh B.R.
      • Lim S.W.
      • Huh J.W.
      • Joo J.K.
      • Kim Y.J.
      • et al.
      Clinical significance of tumor regression grade in rectal cancer with preoperative chemoradiotherapy.
      ,
      • Kim N.K.
      • Baik S.H.
      • Min B.S.
      • Pyo H.R.
      • Choi Y.J.
      • Kim H.
      • et al.
      A comparative study of volumetric analysis, histopathologic downstaging, and tumor regression grade in evaluating tumor response in locally advanced rectal cancer following preoperative chemoradiation.
      ,
      • Xu L.
      • Cai S.
      • Xiao T.
      • Chen Y.
      • Qiu H.
      • Wu B.
      • et al.
      Prognostic significance of tumour regression grade after neoadjuvant chemoradiotherapy for a cohort of patients with locally advanced rectal cancer: an 8-year retrospective single-institutional study.
      ,
      • Reggiani Bonetti L.
      • Lionti S.
      • Domati F.
      • Barresi V.
      Do pathological variables have prognostic significance in rectal adenocarcinoma treated with neoadjuvant chemoradiotherapy and surgery?.
      ,
      • Rullier A.
      • Laurent C.
      • Capdepont M.
      • Vendrely V.
      • Bioulac-Sage P.
      • Rullier E.
      Impact of tumor response on survival after radiochemotherapy in locally advanced rectal carcinoma.
      ].

       Higher local regrowth in advanced tumours

      In a relatively large meta-analysis with individual patient data with 602 patients with a clinical complete response after CRT that were managed by watch and wait there was an increased risk of 37% for local regrowth in cases that were originally staged as cT4 [
      • Chadi S.A.
      • Malcomson L.
      • Ensor J.
      • Riley R.D.
      • Vaccaro C.A.
      • Rossi G.L.
      • et al.
      Factors affecting local regrowth after watch and wait for patients with a clinical complete response following chemoradiotherapy in rectal cancer (InterCoRe consortium): an individual participant data meta-analysis.
      ]. This percentage is similar to the estimated number of cases that show fragmentation as the response mechanism. Local regrowth in smaller tumours (cT1/T2) was significantly lower, 19%. In the five series [
      • Smith F.M.
      • Wiland H.
      • Mace A.
      • Pai R.K.
      • Kalady M.F.
      Depth and lateral spread of microscopic residual rectal cancer after neoadjuvant chemoradiation: implications for treatment decisions.
      ,
      • Perez R.O.
      • Habr-Gama A.
      • Smith F.M.
      • Kosinski L.
      • Sao Juliao G.P.
      • Grzona E.
      • et al.
      Fragmented pattern of tumor regression and lateral intramural spread may influence margin appropriateness after TEM for rectal cancer following neoadjuvant CRT.
      ,
      • Hav M.
      • Libbrecht L.
      • Geboes K.
      • Ferdinande L.
      • Boterberg T.
      • Ceelen W.
      • et al.
      Prognostic value of tumor shrinkage versus fragmentation following radiochemotherapy and surgery for rectal cancer.
      ,
      • Fernandez-Acenero M.J.
      • Estrada Munoz L.
      • Sastre Varela J.
      • Corona Sanchez J.A.
      • Diaz Del Arco C.
      • Garcia Paredes B.
      • et al.
      Prognostic influence of histopathological regression patterns in rectal adenocarcinoma receiving neoadjuvant therapy.
      ,
      • Hayden D.M.
      • Jakate S.
      • Pinzon M.C.
      • Giusto D.
      • Francescatti A.B.
      • Brand M.I.
      • et al.
      Tumor scatter after neoadjuvant therapy for rectal cancer: are we dealing with an invisible margin?.
      ] that describe fragmentation as a response mechanism (total number of patients 460), only limited information is presented about stage in relation to response mechanism. Early stage tumours (cT2) show fragmentation in 25% compared to 44% in cT3 tumours [
      • Perez R.O.
      • Habr-Gama A.
      • Smith F.M.
      • Kosinski L.
      • Sao Juliao G.P.
      • Grzona E.
      • et al.
      Fragmented pattern of tumor regression and lateral intramural spread may influence margin appropriateness after TEM for rectal cancer following neoadjuvant CRT.
      ].

       Margin involvement is more important than TRG and downstaging

      Margin positivity, in particular circumferential resection margin (CRM) involvement is one of the major risk factors for patients with rectal cancer, with an increased risk of both local recurrence as well as distant recurrence after neoadjuvant treatment [
      • Nagtegaal I.D.
      • Quirke P.
      What is the role for the circumferential margin in the modern treatment of rectal cancer?.
      ]. Several studies have compared the prognostic value of CRM involvement with TRG [
      • Gosens M.J.
      • Klaassen R.A.
      • Tan-Go I.
      • Rutten H.J.
      • Martijn H.
      • van den Brule A.J.
      • et al.
      Circumferential margin involvement is the crucial prognostic factor after multimodality treatment in patients with locally advanced rectal carcinoma.
      ,
      • Bouzourene H.
      • Bosman F.T.
      • Matter M.
      • Coucke P.
      Predictive factors in locally advanced rectal cancer treated with preoperative hyperfractionated and accelerated radiotherapy.
      ,
      • Machiels J.P.
      • Aydin S.
      • Bonny M.A.
      • Hammouch F.
      • Sempoux C.
      What is the best way to predict disease-free survival after preoperative radiochemotherapy for rectal cancer patients: tumor regression grading, nodal status, or circumferential resection margin invasion?.
      ], and all multivariable analyses pointed towards CRM as the main prognostic factor over TRG, but also compared to downstaging [
      • Mawdsley S.
      • Glynne-Jones R.
      • Grainger J.
      • Richman P.
      • Makris A.
      • Harrison M.
      • et al.
      Can histopathologic assessment of circumferential margin after preoperative pelvic chemoradiotherapy for T3–T4 rectal cancer predict for 3-year disease-free survival?.
      ]. Interestingly, in the presence of fragmentation as reaction pattern, it has been suggested that negative margins might be no longer informative [
      • Smith F.M.
      • Wiland H.
      • Mace A.
      • Pai R.K.
      • Kalady M.F.
      Depth and lateral spread of microscopic residual rectal cancer after neoadjuvant chemoradiation: implications for treatment decisions.
      ], due to the discontinuity of residual tumour spread. Larger studies are necessary to confirm this hypothesis.

       Negative biopsies do not rule out residual tumour

      A number of studies have tried to determine the presence of residual tumour with posttreatment biopsies (Table 1). In total 351 cases have been described, from Brazil [
      • Perez R.O.
      • Habr-Gama A.
      • Pereira G.V.
      • Lynn P.B.
      • Alves P.A.
      • Proscurshim I.
      • et al.
      Role of biopsies in patients with residual rectal cancer following neoadjuvant chemoradiation after downsizing: can they rule out persisting cancer?.
      ], China [
      • Xiao L.
      • Yu X.
      • Deng W.
      • Feng H.
      • Chang H.
      • Xiao W.
      • et al.
      Pathological assessment of rectal cancer after neoadjuvant chemoradiotherapy: distribution of residual cancer cells and accuracy of biopsy.
      ], Taiwan [
      • Kuo L.J.
      • Chiou J.F.
      • Tai C.J.
      • Chang C.C.
      • Kung C.H.
      • Lin S.E.
      • et al.
      Can we predict pathologic complete response before surgery for locally advanced rectal cancer treated with preoperative chemoradiation therapy?.
      ] and Spain [
      • Lopez-Lopez V.
      • Abrisqueta J.
      • Lujan J.
      • Hernandez Q.
      • Ono A.
      • Parrilla P.
      Utility of rectoscopy in the assessment of response to neoadjuvant treatment for locally advanced rectal cancer.
      ]. While both the specificity and the negative predictive value are high, the overall accuracy of the biopsies is only 51.3%, and with a positive predictive value of 26.7% the impact of negative biopsies is low. Based on the data of Fig. 2 (with 54% of ypT2-4 patients presenting with a tumour-free submucosa), this was expected. However, even in cases that appear clinically benign [
      • Rupinski M.
      • Szczepkowski M.
      • Malinowska M.
      • Mroz A.
      • Pietrzak L.
      • Wyrwicz L.
      • et al.
      Watch and wait policy after preoperative radiotherapy for rectal cancer; management of residual lesions that appear clinically benign.
      ] with an initial benign biopsy, still 30% of cases turned out to be malignant in a Polish series of only 7 cases. In cases with residual ulceration as the only macroscopic sign, only 27% show a complete pathological response [
      • Smith F.M.
      • Wiland H.
      • Mace A.
      • Pai R.K.
      • Kalady M.F.
      Depth and lateral spread of microscopic residual rectal cancer after neoadjuvant chemoradiation: implications for treatment decisions.
      ].
      Table 1Relevance of postoperative biopsies for detection of complete pathological response (pCR). PPV: positive predictive value, NPV: negative predictive value.
      AuthornNegative biopsies
      npCRSpecificitySensitivityPPVNPVAccuracy
      Perez
      • Chow O.S.
      • Patil S.
      • Keskin M.
      • Smith J.J.
      • Widmar M.
      • Smith D.D.
      • et al.
      Variation in the thoroughness of pathologic assessment and response rates of locally advanced rectal cancers after chemoradiation.
      39143100.0%69.4%21.4%100.0%71.8%
      Xiao
      • Greenbaum A.
      • Martin D.R.
      • Bocklage T.
      • Lee J.H.
      • Ness S.A.
      • Rajput A.
      Tumor heterogeneity as a predictor of response to neoadjuvant chemotherapy in locally advanced rectal cancer.
      79661694.1%19.4%24.2%92.0%35.9%
      Kuo
      • Yeom S.S.
      • Lee S.Y.
      • Kim C.H.
      • Kim Y.J.
      • Nam T.K.
      • Kim H.R.
      Non-operative treatment outcome for rectal cancer patient with clinical complete response after neoadjuvant chemoradiotherapy.
      16611725100.0%34.7%21.4%100.0%44.3%
      Lopez-Lopez
      • Sorenson E.
      • Lambreton F.
      • Yu J.Q.
      • Li T.
      • Denlinger C.S.
      • Meyer J.E.
      • et al.
      Impact of PET/CT for restaging patients with locally advanced rectal cancer after neoadjuvant chemoradiation.
      673518100.0%65.3%51.4%100.0%74.6%
      Total3512326298.4%41.0%26.7%99.0%51.3%

       Near complete response is associated with a poor prognosis

      Following the steps of TRG, one would expect that a near complete response is almost as good as it gets, only limited numbers of tumour cells remain in the resection specimen and this would result in a good outcome. However, in patients with locally advanced rectal cancer, this group in particular presents with an unexpected poor outcome [
      • Swellengrebel H.A.
      • Bosch S.L.
      • Cats A.
      • Vincent A.D.
      • Dewit L.G.
      • Verwaal V.J.
      • et al.
      Tumour regression grading after chemoradiotherapy for locally advanced rectal cancer: a near pathologic complete response does not translate into good clinical outcome.
      ,
      • Kim J.Y.
      • Park I.J.
      • Hong S.M.
      • Lee J.L.
      • Yoon Y.S.
      • Kim C.W.
      • et al.
      Is pathologic near-total regression an appropriate indicator of a good response to preoperative chemoradiotherapy based on oncologic outcome of disease?.
      ]. In these studies, near complete response was defined as isolated tumour cells or small groups of tumour cells in fibrotic stroma, that are difficult to find, corresponding to Dworak grade 3. Survival in this group, after TME surgery, was significantly worse than in the pCR group, which was also confirmed in a meta-analysis [
      • Kim J.Y.
      • Park I.J.
      • Hong S.M.
      • Lee J.L.
      • Yoon Y.S.
      • Kim C.W.
      • et al.
      Is pathologic near-total regression an appropriate indicator of a good response to preoperative chemoradiotherapy based on oncologic outcome of disease?.
      ], but also worse compared to the group with minimal response [
      • Swellengrebel H.A.
      • Bosch S.L.
      • Cats A.
      • Vincent A.D.
      • Dewit L.G.
      • Verwaal V.J.
      • et al.
      Tumour regression grading after chemoradiotherapy for locally advanced rectal cancer: a near pathologic complete response does not translate into good clinical outcome.
      ].
      The simple explanation is that this is a form of extensive fragmentation, and that these cases have actually a higher tumour stage compared to those with less obvious histological regression, i.e. Dworak grade 2, where shrinkage is the main form of regression. This is in line with findings of Fernandez-Acenero [
      • Fernandez-Acenero M.J.
      • Estrada Munoz L.
      • Sastre Varela J.
      • Corona Sanchez J.A.
      • Diaz Del Arco C.
      • Garcia Paredes B.
      • et al.
      Prognostic influence of histopathological regression patterns in rectal adenocarcinoma receiving neoadjuvant therapy.
      ], who showed that fragmentation was indeed the main pattern of response in the near-complete response group (58%), and that the presence of fragmentation was linked to the absence of downstaging. We can only hypothesize about the more complex interpretation of this reaction pattern and the poor outcome.
      In untreated patients, the role of tumour budding (i.e. single tumour cells and small groups of tumour cells) is associated with a poor outcome in general [
      • Rogers A.C.
      • Winter D.C.
      • Heeney A.
      • Gibbons D.
      • Lugli A.
      • Puppa G.
      • et al.
      Systematic review and meta-analysis of the impact of tumour budding in colorectal cancer.
      ] and a lack of response to neoadjuvant therapy [
      • Rogers A.C.
      • Gibbons D.
      • Hanly A.M.
      • Hyland J.M.
      • O’Connell P.R.
      • Winter D.C.
      Prognostic significance of tumor budding in rectal cancer biopsies before neoadjuvant therapy.
      ]. Tumour buds are considered a feature of epithelial-mesenchymal transition (EMT), and as such, is associated with the mesenchymal subtype [
      • Trinh A.
      • Ladrach C.
      • Dawson H.E.
      • Ten Hoorn S.
      • Kuppen P.J.K.
      • Reimers M.S.
      • et al.
      Tumour budding is associated with the mesenchymal colon cancer subtype and RAS/RAF mutations: a study of 1320 colorectal cancers with Consensus Molecular Subgroup (CMS) data.
      ] that is associated with a poor outcome. The induction of an EMT-like phenotype in colorectal cancer due to radiation [
      • de Marcondes P.G.
      • Morgado-Diaz J.A.
      The role of EphA4 signaling in radiation-induced EMT-like phenotype in colorectal cancer cells.
      ,
      • Diao W.L.
      • Luo L.
      • Luo Q.
      Effect of X-ray irradiation on epithelial-mesenchymal transition of colorectal cancer SW480 cells.
      ] and chemotherapy [
      • Trumpi K.
      • Ubink I.
      • Trinh A.
      • Djafarihamedani M.
      • Jongen J.M.
      • Govaert K.M.
      • et al.
      Neoadjuvant chemotherapy affects molecular classification of colorectal tumors.
      ] have been described before. The molecular features of EMT, such as reduced E-cadherin and mir200c, have been shown to be associated with decreased response to CRT as well as poor outcome in rectal cancer patients [

      Bhangu A, Wood G, Brown G, Darzi A, Tekkis P, Goldin R. The role of epithelial mesenchymal transition and resistance to neoadjuvant therapy in locally advanced rectal cancer. Colorectal Dis: Off J Assoc Coloproctol Great Britain Ireland 2014;16:O133–43.

      ]. It is not unlikely to assume that the biology behind fragmentation involves EMT.
      Alternatively, the tumour-stroma ratio (TSR) has been identified as a prognostic factor in untreated patients. The presence of extensive stroma between groups of tumour cells is associated with a poor outcome [
      • Park J.H.
      • Richards C.H.
      • McMillan D.C.
      • Horgan P.G.
      • Roxburgh C.S.
      The relationship between tumour stroma percentage, the tumour microenvironment and survival in patients with primary operable colorectal cancer.
      ]. We have shown before that neoadjuvant therapy increased the amount of tumour stroma [
      • Nagtegaal I.D.
      • Marijnen C.A.
      • Kranenbarg E.K.
      • Mulder-Stapel A.
      • Hermans J.
      • van de Velde C.J.
      • et al.
      Short-term preoperative radiotherapy interferes with the determination of pathological parameters in rectal cancer.
      ], even after short-term radiotherapy. After neoadjuvant chemotherapy, increased stroma (low TSR) or fibrosis is also associated with disease recurrence [
      • Hansen T.F.
      • Kjaer-Frifeldt S.
      • Lindebjerg J.
      • Rafaelsen S.R.
      • Jensen L.H.
      • Jakobsen A.
      • et al.
      Tumor-stroma ratio predicts recurrence in patients with colon cancer treated with neoadjuvant chemotherapy.
      ]. It is likely that increased amounts of stroma are relevant in fragmentation.

       Tumour deposits after neoadjuvant therapy are different

      A recent meta-analysis focused on tumour deposits (TD) after neoadjuvant therapy [
      • Lord A.C.
      • Graham Martinez C.
      • D'Souza N.
      • Pucher P.H.
      • Brown G.
      • Nagtegaal I.D.
      The significance of tumour deposits in rectal cancer after neoadjuvant therapy: a systematic review and meta-analysis.
      ]. The mean prevalence of TD (24.5% of patients) was only slightly increased compared to non-pretreated colorectal cancer patients (22.5%) [
      • Nagtegaal I.D.
      • Knijn N.
      • Hugen N.
      • Marshall H.C.
      • Sugihara K.
      • Tot T.
      • et al.
      Tumor deposits in colorectal cancer: improving the value of modern staging – a systematic review and meta-analysis.
      ], but compared to the cases with rectal carcinoma without neoadjuvant therapy (33.6%) [
      • Harrison J.C.
      • Dean P.J.
      • el Zeky F.
      • Vander Z.R.
      From Dukes through Jass: pathological prognostic indicators in rectal cancer.
      ,
      • Ueno H.
      • Mochizuki H.
      Clinical significance of extrabowel skipped cancer infiltration in rectal cancer.
      ,
      • Shimada Y.
      • Takii Y.
      Clinical impact of mesorectal extranodal cancer tissue in rectal cancer: detailed pathological assessment using whole-mount sections.
      ] there is a decreased number. Moreover, the established strong relations of TD with lymphatic invasion, extramural vascular invasion and perineural invasion were no longer present in the neoadjuvant setting, suggesting that at least part of these TD may have a different origin, and may indeed form part of the fragmentation pattern.

      Conclusions

      With the increasing importance of neoadjuvant treatment for rectal cancer, there is an urgent need for relevant biomarkers that predict therapy response and even more important predict recurrent disease. At the moment, these are lacking. Response categorization relies mainly on two different strategies: the presence of downstaging and TRG. The inherent subjectivity of TRG limits its implementation. Downstaging does not acknowledge the heterogeneity of tumour response. The presence of residual lymph node metastases after complete response of the primary tumour illustrates this point.
      The recognition of different response patterns explains a number of clinical conundrums, including discrepancies in risk of residual tumour and regrowth. The recognition of the different patterns of response explains the high regrowth observed in the more advanced rectal cancers. Early recognition of patients at risk would improve outcomes and potentially allow us to adjust treatment regimens. Larger studies are required to define the prognostic relevance in different groups of tumours, including small and advanced rectal cancer.

      Future directions

      Recognition of different patterns of response can provide us with the essential next step for treatment of patients with rectal cancer. For years, biomarker studies have been performed in order to find the holy grail: the biomarker that predicts therapy response. If such a biomarker would exist, it would provide us with guidance for therapeutic decisions. Up to now, results are disappointing, with no biomarkers implemented in clinical practice [
      • Perez R.O.
      • Habr-Gama A.
      • Sao Juliao G.P.
      • Vailati B.B.
      • Fernandez L.M.
      • Gama-Rodrigues J.
      • et al.
      Should we give up the search for a clinically useful gene signature for the prediction of response of rectal cancer to neoadjuvant chemoradiation?.
      ]. In order to predict benefits for individual patients, biomarkers are essential. However, most studies were focused on complete response, mainly determined 6 weeks after treatment.
      In the current era, priorities are changing, with increased application of local excision and watchful waiting strategies. Local excision would benefit patients with a non-fragmenting pattern of response, in particular those patients that have a small tumour on pre-treatment imaging, where fragmentation is less likely to occur. However, if a pattern of fragmentation is present, a resection will still be necessary. Since it is likely that differences in patterns of response are based on differences in the underlying biology of the tumour, analysis of biomarker studies with pattern as outcome might finally deliver these necessary biomarkers. Moreover, different treatment regimens might result in different patterns of response, which would further personalize treatment.
      The increased focus on microenvironmental interactions of tumour cells offers further opportunities to understand the biology of tumour response. EMT might be involved in the biology of tumour response. The morphological resemblance of tumour fragments and tumour buds suggests that this might be relevant. The relations between CRT and EMT have been described above [
      • de Marcondes P.G.
      • Morgado-Diaz J.A.
      The role of EphA4 signaling in radiation-induced EMT-like phenotype in colorectal cancer cells.
      ,
      • Diao W.L.
      • Luo L.
      • Luo Q.
      Effect of X-ray irradiation on epithelial-mesenchymal transition of colorectal cancer SW480 cells.
      ,
      • Trumpi K.
      • Ubink I.
      • Trinh A.
      • Djafarihamedani M.
      • Jongen J.M.
      • Govaert K.M.
      • et al.
      Neoadjuvant chemotherapy affects molecular classification of colorectal tumors.
      ,

      Bhangu A, Wood G, Brown G, Darzi A, Tekkis P, Goldin R. The role of epithelial mesenchymal transition and resistance to neoadjuvant therapy in locally advanced rectal cancer. Colorectal Dis: Off J Assoc Coloproctol Great Britain Ireland 2014;16:O133–43.

      ]. Moreover, processes such as radiation-induced inflammation, combined with tumour-specific immune responses may be instrumental in causing different response patterns. The comparison with response of solid tumours after neoadjuvant immunotherapy might be relevant [
      • Cottrell T.R.
      • Thompson E.D.
      • Forde P.M.
      • Stein J.E.
      • Duffield A.S.
      • Anagnostou V.
      • et al.
      Pathologic features of response to neoadjuvant anti-PD-1 in resected non-small-cell lung carcinoma: a proposal for quantitative immune-related pathologic response criteria (irPRC).
      ], since there are common reaction patterns observed.

      Declaration of Competing Interest

      The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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